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CONTRIBUTION OF SCLEROTOME TO THE EMBRYONIC HEMATOPOIETIC STEM CELL NICHE AND VASCULAR SMOOTH MUSCLE
Poster Presentations / Experimental Hematology 2019;76 (Suppl):S52−S96
3066 - ZBTB11 IS REQUIRED FOR HEMATOPOIETIC
S71
3068 - PROLIFERATION, DNA DAMAGE RESPONSE AND
STEM CELL FUNCTION M. Cristina Keightley1,2, Huimin Cao3, Joly Kwek4 , Ben Cao3 , Susie Nilsson3, Graham Lieschke4 1 Dept of Pharmacy and Biomedical Sciences, La Trobe University, Flora Hill, Australia; 2Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Flora Hill, Australia; 3Biomedical Manufacturing, CSIRO, Clayton, Australia; 41Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
HEMATOPOIETIC STEM CELL FUNCTION: A DYNAMIC, CONTEXT DEPENDENT RELATION Satish Khurana1, Atreyi Biswas1, Irene Roy1, Prathibha Babu1, Javed Manesia2, Sarah Schouteden3, Joerg Huelsken4, Adam Lacy-Hulbert5, Catherine Verfaillie3, Satish Khurana1 1 IISER TVM, Thiruvananthapuram, India; 2Canadian Blood Services, Ottawa, Canada; 3KU Leuven, Leuven, Belgium; 4EPFL, Lausanne, Switzerland; 5Benaroya Research Institute, Seattle, United States
Hematopoiesis orchestrates the daily requirement of producing and maintaining the various blood cell lineages in the right quantities. It must also respond rapidly to injury and infection to expand specific lineages on demand then return them to steady state levels. Understanding the sophisticated mechanisms regulating this balancing act can uncover therapeutic targets for manipulating blood cell production in disease treatment. In a forward genetic screen for zebrafish myeloid mutants, we identified a requirement for ZBTB11, a ZBTB (BTB/POZ) transcriptional repressor, in definitive hematopoiesis. To understand its relevance to mammalian systems, we extended these studies to mouse. When Zbtb11 is deleted in the hematopoietic compartment embryos do not survive past E18.5 likely due to insufficient hematopoiesis. Phenotypic HSCs (SLAM) are specified in over-abundance at E14.5 through E17.5 compared to controls. Functional analysis, however, shows they are unable to effectively differentiate into committed progenitors and mature lineages in fetal liver. Hematopoietic content in fetal bone marrow is mostly absent at E16.5 − E17.5 pointing to bone marrow failure. Further analysis showed Zbtb11-/- HSCs fail to proliferate in an in vitro proliferation assay and are constrained in their progression through the cell cycle demonstrating a role for Zbtb11 in proliferation and cell cycle regulation in mammalian hematopoiesis. We have undertaken scRNAseq in Zbtb11 /- and control HSC populations to understand heterogeneity within and between these populations, and what genetic programs have been altered. Mechanistic evaluation is under way. In summary, our findings establish a conserved cell autonomous role for Zbtb11 in mammalian definitive hematopoiesis, specifically in HSC function, opening up new avenues for understanding and manipulating HSCs.
Proliferation in hematopoietic stem cells (HSCs) has been linked to stemness. A variety of mouse transgenic lines with increased HSC proliferation rates have shown concomitant deterioration of their function (Dev. Dyn. 2016). While some studies implicated DNA damage accumulation as the underlying reason (Walter D. et al. in Nature 2015), others have maintained that, in fact, quiescence of primitive HSCs might be the reason behind poor resolution of DNA damages (Mohrin M et al. in Cell Stem Cell 2010). In our earlier studies, we demonstrated that outside-in integrin signaling is important to maintain HSC quiescence (Khurana S. et al. in Nat. Comm. 2016). We showed loss of HSC function in mice deficient in interaction between Integrin-av (Itgav; Vav-Itgav-/- mice) and its ligand Periostin (Postn; Postn-/- mice). Our recent studies show that increase in proliferation of fetal liver (FL) HSCs in these mice results in their efficient expansion without any loss of function. Postn deficient FL tissues showed clear increase in the frequency of primitive HSCs with long-term engraftment potential. They proliferated faster, without any functional decline. However, Vav-Itgav-/- FL HSCs transiting from proliferative to quiescent phenotype showed dependence on Itgav-Postn interaction for quiescence and functional integrity. On the basis of RNASeq based differential pathway analysis using BM and E14.5 FL derived HSCs, we hypothesized that better DNA damage response (DDR) pathways in FL derived HSCs could result in these observations. In deed we observed that HSCs derived from FL show better resistance to DNA damage than adult bone marrow. We went on to show the importance of integrin ligand Postn in the creation of hematopoietic niche in FL tissue. Overall, we propose through our results that proliferation in HSCs affects HSC function in developmental context dependent manner.
3067 - CONTRIBUTION OF SCLEROTOME TO THE
3069 - ENVIRONMENTAL OPTIMIZATION ENABLES
EMBRYONIC HEMATOPOIETIC STEM CELL NICHE AND VASCULAR SMOOTH MUSCLE Clair Kelley1 , Wilson Clements1 , Nicole Glenn1, Massimo Santoro2 1 St. Jude Children’s Research Hospital, Memphis, United States; 2 University of Padua, Italy, Padua, Italy
MAINTENANCE OF QUIESCENT HEMATOPOIETIC STEM CELLS EX VIVO Hiroshi Kobayashi1,2, Keiyo Takubo2 1 Department of Stem Cell Biology, Shinjuku-ku, Japan; 2National Center for Global Health and Medicine, Shinjuku-ku, Japan
Hematopoietic stem cells (HSCs) are a self-renewing population that sustains production of blood lineages throughout life. Vertebrate HSCs are first specified in the embryo, arising from hemogenic endothelium in the ventral wall of the dorsal aorta (DA). The cell types within this three-dimensional environment and the signals they provide to promote HSC development are not well defined. A better understanding of native embryonic HSC specification niche could inform efficient directed differentiation of true HSCs from induced pluripotent stem cells for clinical and research applications. We previously showed that wnt16 is required for development of zebrafish HSCs, as well as normal patterning of the ventral somite, the sclerotome. We hypothesized that normal sclerotome patterning is required for HSC specification by contributing to the cellular environment that presents inductive signals. To follow sclerotome development in living embryos, we developed transgenic reporter zebrafish where fluorophore expression is driven by regulatory elements from the sclerotome gene, pax1a. We show that sclerotome-derived cells migrate to and make contact with hemogenic endothelium of the DA shortly before initiation of the definitive hematopoietic program, demonstrating that they are present at the right time and place to present specification signals. By live imaging and profiling of sorted cells, we show these cells also give rise to arterial vascular smooth muscle cells. Loss-of-function analyses demonstrate a requirement for multiple sclerotome genes, for both patterning of sclerotome, and subsequent specification of HSCs and downstream hematopoietic lineages. Our data indicate that sclerotomal cells contribute to the HSC specification niche of the early embryo, and form a basis for defining the niche architecture and signals that drive definitive hematopoietic programming.
Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of steady state HSCs and test effects of defined factors in vitro under nearphysiological conditions.
3066 - ZBTB11 IS REQUIRED FOR HEMATOPOIETIC
S71
3068 - PROLIFERATION, DNA DAMAGE RESPONSE AND
STEM CELL FUNCTION M. Cristina Keightley1,2, Huimin Cao3, Joly Kwek4 , Ben Cao3 , Susie Nilsson3, Graham Lieschke4 1 Dept of Pharmacy and Biomedical Sciences, La Trobe University, Flora Hill, Australia; 2Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Flora Hill, Australia; 3Biomedical Manufacturing, CSIRO, Clayton, Australia; 41Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
HEMATOPOIETIC STEM CELL FUNCTION: A DYNAMIC, CONTEXT DEPENDENT RELATION Satish Khurana1, Atreyi Biswas1, Irene Roy1, Prathibha Babu1, Javed Manesia2, Sarah Schouteden3, Joerg Huelsken4, Adam Lacy-Hulbert5, Catherine Verfaillie3, Satish Khurana1 1 IISER TVM, Thiruvananthapuram, India; 2Canadian Blood Services, Ottawa, Canada; 3KU Leuven, Leuven, Belgium; 4EPFL, Lausanne, Switzerland; 5Benaroya Research Institute, Seattle, United States
Hematopoiesis orchestrates the daily requirement of producing and maintaining the various blood cell lineages in the right quantities. It must also respond rapidly to injury and infection to expand specific lineages on demand then return them to steady state levels. Understanding the sophisticated mechanisms regulating this balancing act can uncover therapeutic targets for manipulating blood cell production in disease treatment. In a forward genetic screen for zebrafish myeloid mutants, we identified a requirement for ZBTB11, a ZBTB (BTB/POZ) transcriptional repressor, in definitive hematopoiesis. To understand its relevance to mammalian systems, we extended these studies to mouse. When Zbtb11 is deleted in the hematopoietic compartment embryos do not survive past E18.5 likely due to insufficient hematopoiesis. Phenotypic HSCs (SLAM) are specified in over-abundance at E14.5 through E17.5 compared to controls. Functional analysis, however, shows they are unable to effectively differentiate into committed progenitors and mature lineages in fetal liver. Hematopoietic content in fetal bone marrow is mostly absent at E16.5 − E17.5 pointing to bone marrow failure. Further analysis showed Zbtb11-/- HSCs fail to proliferate in an in vitro proliferation assay and are constrained in their progression through the cell cycle demonstrating a role for Zbtb11 in proliferation and cell cycle regulation in mammalian hematopoiesis. We have undertaken scRNAseq in Zbtb11 /- and control HSC populations to understand heterogeneity within and between these populations, and what genetic programs have been altered. Mechanistic evaluation is under way. In summary, our findings establish a conserved cell autonomous role for Zbtb11 in mammalian definitive hematopoiesis, specifically in HSC function, opening up new avenues for understanding and manipulating HSCs.
Proliferation in hematopoietic stem cells (HSCs) has been linked to stemness. A variety of mouse transgenic lines with increased HSC proliferation rates have shown concomitant deterioration of their function (Dev. Dyn. 2016). While some studies implicated DNA damage accumulation as the underlying reason (Walter D. et al. in Nature 2015), others have maintained that, in fact, quiescence of primitive HSCs might be the reason behind poor resolution of DNA damages (Mohrin M et al. in Cell Stem Cell 2010). In our earlier studies, we demonstrated that outside-in integrin signaling is important to maintain HSC quiescence (Khurana S. et al. in Nat. Comm. 2016). We showed loss of HSC function in mice deficient in interaction between Integrin-av (Itgav; Vav-Itgav-/- mice) and its ligand Periostin (Postn; Postn-/- mice). Our recent studies show that increase in proliferation of fetal liver (FL) HSCs in these mice results in their efficient expansion without any loss of function. Postn deficient FL tissues showed clear increase in the frequency of primitive HSCs with long-term engraftment potential. They proliferated faster, without any functional decline. However, Vav-Itgav-/- FL HSCs transiting from proliferative to quiescent phenotype showed dependence on Itgav-Postn interaction for quiescence and functional integrity. On the basis of RNASeq based differential pathway analysis using BM and E14.5 FL derived HSCs, we hypothesized that better DNA damage response (DDR) pathways in FL derived HSCs could result in these observations. In deed we observed that HSCs derived from FL show better resistance to DNA damage than adult bone marrow. We went on to show the importance of integrin ligand Postn in the creation of hematopoietic niche in FL tissue. Overall, we propose through our results that proliferation in HSCs affects HSC function in developmental context dependent manner.
3067 - CONTRIBUTION OF SCLEROTOME TO THE
3069 - ENVIRONMENTAL OPTIMIZATION ENABLES
EMBRYONIC HEMATOPOIETIC STEM CELL NICHE AND VASCULAR SMOOTH MUSCLE Clair Kelley1 , Wilson Clements1 , Nicole Glenn1, Massimo Santoro2 1 St. Jude Children’s Research Hospital, Memphis, United States; 2 University of Padua, Italy, Padua, Italy
MAINTENANCE OF QUIESCENT HEMATOPOIETIC STEM CELLS EX VIVO Hiroshi Kobayashi1,2, Keiyo Takubo2 1 Department of Stem Cell Biology, Shinjuku-ku, Japan; 2National Center for Global Health and Medicine, Shinjuku-ku, Japan
Hematopoietic stem cells (HSCs) are a self-renewing population that sustains production of blood lineages throughout life. Vertebrate HSCs are first specified in the embryo, arising from hemogenic endothelium in the ventral wall of the dorsal aorta (DA). The cell types within this three-dimensional environment and the signals they provide to promote HSC development are not well defined. A better understanding of native embryonic HSC specification niche could inform efficient directed differentiation of true HSCs from induced pluripotent stem cells for clinical and research applications. We previously showed that wnt16 is required for development of zebrafish HSCs, as well as normal patterning of the ventral somite, the sclerotome. We hypothesized that normal sclerotome patterning is required for HSC specification by contributing to the cellular environment that presents inductive signals. To follow sclerotome development in living embryos, we developed transgenic reporter zebrafish where fluorophore expression is driven by regulatory elements from the sclerotome gene, pax1a. We show that sclerotome-derived cells migrate to and make contact with hemogenic endothelium of the DA shortly before initiation of the definitive hematopoietic program, demonstrating that they are present at the right time and place to present specification signals. By live imaging and profiling of sorted cells, we show these cells also give rise to arterial vascular smooth muscle cells. Loss-of-function analyses demonstrate a requirement for multiple sclerotome genes, for both patterning of sclerotome, and subsequent specification of HSCs and downstream hematopoietic lineages. Our data indicate that sclerotomal cells contribute to the HSC specification niche of the early embryo, and form a basis for defining the niche architecture and signals that drive definitive hematopoietic programming.
Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of steady state HSCs and test effects of defined factors in vitro under nearphysiological conditions.